The availability of Tetrahymena pre-rRNA of discrete size, produced by transcription of recombinant plasmids with bacteriophage SP6 RNA polymerase, has permitted a more detailed investigation of the self-splicing reaction. The predicted splicing intermediate, the product of cleavage by guanosine at the 5′ splice site, was identified. This intermediate was tested in the intermolecular exon ligation reaction and found to be competent to undergo the second step of splicing. These results and others that evaluated the reactivity of the 5′ and 3′ splice sites independently show that splicing occurs in two separable steps. The 3′ splice site was found to be susceptible to site-specific hydrolysis leaving a 3′ hydroxyl terminus. This is interpreted as an indication that the 3′ splice site is activated for nucleophilic attack in general and for exon ligation in particular. Preliminary evidence for specific hydrolysis at the 5′ splice site was also obtained. All of the newly characterized intervening sequence RNA-mediated reactions as well as those found previously are divided into three categories: (1) transesterification by guanosine at sites following two or three pyrimidine nucleotides (and, as a minor reaction, at sites following other guanosine residues); (2) transesterification by oligopyrimidines or by the 5′ exon (which terminates with C-U-C-U-C-U_OH) at the site following the 3′-terminal guanosine residue of the intervening sequence; and (3) specific hydrolysis at the splice sites.

One of the products of the reactions at the 3′ splice site is a molecule that contains the 5′ exon still attached to the intervening sequence. It has a 3′-terminal G_OH and undergoes cyclization both at the normal cyclization site within the intervening sequence and at the 5′ splice site. The finding that the splice site can act as a cyclization site, combined with the earlier observation that the normal cyclization site is subject to attack by guanosine mononucleotide, leads us to propose that all these reactions may be occurring in the same active site. Translocation (a conformational change) would then bring different oligopyrimidine sequences into the active site for attack by guanosine. On the basis of the experimental results, a model for the local structure at the active site is described. A key feature of the model is the interaction between the U at the end of the oligopyrimidine sequence, a G residue within the internal guide sequence in the intervening sequence, and another G residue that can be either the attacking group for transesterification or the 3′-terminal G of the intervening sequence.